Peptides are easily denatured due to their low stability, degraded by in-vivo proteases, and excreted through the kidney due to their relatively small size. Accordingly, in order to maintain a specific concentration in the blood of a peptide drug in its active form, it is necessary to administer the peptide drug frequently to a patient. However, peptide drugs are usually administered in the form of injectable preparations, and such frequent administration cause severe discomfort for patients. To solve such problem, there have been developed a number of methods, e.g., a method for transferring the peptide drug through oropharyngeal or nasopharyngeal inhalation by increasing the permeation of the peptide drug through the biological membranes, a method for modifying a specific amino acid sequence which is sensitive to proteases (e.g., GLP-1 amino acid sequence for preventing loss of the titers by a dipeptidyl peptidase) in order to stabilize the peptide by inhibiting the degradation by the enzyme, and a method for chemically adding a non-peptidyl polymer with a high solubility, such as polyethylene glycol (PEG), on the surface of the peptide.
PEG, which has been used as one of non-peptidyl polymers, non-specifically binds to a specific site or multiple sites of a target peptide to attain the effect of increasing the molecular weight of the peptide, the resulting PEG-peptide resists the loss through the kidney and enzymatic hydrolysis, without causing any side-effects. For example, International Pat. Publication No. WO 2006/076471 describes sustaining the physiological activity of a B-type natriuretic peptide (BNP) used as congestive heart failure therapeutic agent by binding PEG thereto, and U.S. Pat. No. 6,924,264 describes increasing the in-vivo residence time of an exendin-4 drug by way of binding PEG to the lysine residue thereof.
These methods prolong the in-vivo residence time of a peptide drug by increasing the molecular weight of PEG, but as the molecular weight increases, the titer of the peptide drug becomes significantly reduced. In addition, the non-specific binding of PEG may shield the active domain of a physiologically active polypeptide to significantly lower the activity of the polypeptide.
Therefore, there is a need to develop an improved method for preparing a conjugate of a physiologically active polypeptide and a non-peptidyl polymer, in which the polymer is linked to the peptide in a site-specific manner that does not affect the polypeptide's activity.
The present inventors have completed the invention by confirming that a physiologically active polypeptide conjugate having a non-peptidyl polymer site-specifically linked can be prepared in a high yield by adjusting the pH and the alcohol content of the reaction medium.